Treatment of ocular disease

ABSTRACT

Disclosed are methods for treating eye diseases or conditions characterized by vascular instability, vascular leakage and neovacularization such as diabetic macular edema, age-related macular edema, choroidal neovascularization, diabetic retinopathy, trauma, ocular ischemia, retinal angiomatous proliferation, macular telangiectasia and uveitis.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority U.S. Provisional Application Ser. No.61/546,708 filed Oct. 13, 2011. The entire content of U.S. ProvisionalApplication Ser. No. 61/546,708 is incorporated herein by reference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablesequence listing submitted concurrently herewith and identified asfollows: One 92 KB ASCII (Text) file named“233106-332748_Seq_Listing_ST25.txt,” created on Oct. 12, 2012, at 3:07pm.

FIELD

Methods for treating eye diseases or conditions characterized byvascular instability, vascular leakage, and neovacularization such asocular edema, ocular neovascularization, diabetic macular edema,age-related macular degeneration, choroidal neovascularization, diabeticretinopathy, retinal vein occlusion (central or branch), ocularischemia, ocular trauma, surgery induced edema, and uveitis.

BACKGROUND

The eye comprises several structurally and functionally distinctvascular beds, which supply ocular components critical to themaintenance of vision. These include the retinal and choroidalvasculatures, which supply the inner and outer portions of the retina,respectively, and the limbal vasculature located at the periphery of thecornea. Injuries and diseases that impair the normal structure orfunction of these vascular beds are among the leading causes of visualimpairment and blindness. For example, diabetic retinopathy is the mostcommon disease affecting the retinal vasculature, and is the leadingcause of vision loss among the working age population in the UnitedStates. Vascularization of the cornea secondary to injury or disease isyet another category of ocular vascular disease that can lead to severeimpairment of vision.

“Macular degeneration” is a general medical term that applies to any ofseveral disease syndromes, which involve a gradual loss or impairment ofeyesight due to cell and tissue degeneration of the yellow macularregion in the center of the retina. Macular degeneration is oftencharacterized as one of two types, non-exudative (dry form) or exudative(wet form). Although both types are bilateral and progressive, each typemay reflect different pathological processes. The wet form ofage-related macular degeneration (AMD) is the most common form ofchoroidal neovascularization and a leading cause of blindness in theelderly. AMD affects millions of Americans over the age of 60, and isthe leading cause of new blindness among the elderly.

Choroidal neovascular membrane (CNVM) is a problem that is related to awide variety of retinal diseases, but is most commonly linked toage-related macular degeneration. With CNVM, abnormal blood vesselsstemming from the choroid (the blood vessel-rich tissue layer justbeneath the retina) grow up through the retinal layers. These newvessels are very fragile and break easily, causing blood and fluid topool within the layers of the retina.

Diabetes (diabetes mellitus) is a metabolic disease caused by theinability of the pancreas to produce insulin or to use the insulin thatis produced. The most common types of diabetes are type 1 diabetes(often referred to as Juvenile Onset Diabetes Mellitus) and type 2diabetes (often referred to as Adult Onset Diabetes Mellitus). Type 1diabetes results from the body's failure to produce insulin due to lossof insulin producing cells, and presently requires the person to injectinsulin. Type 2 diabetes generally results from insulin resistance, acondition in which cells fail to use insulin properly. Type 2 diabetesmay have a component of insulin deficiency as well.

Diabetes is directly responsible for a large number of diseaseconditions, including conditions or diseases of the eye includingdiabetic retinopathy (DR) and diabetic macular edema (DME) which areleading causes of vision loss and blindness in most developed countries.The increasing number of individuals with diabetes worldwide suggeststhat DR and DME will continue to be major contributors to vision lossand associated functional impairment for years to come.

Diabetic retinopathy is a complication of diabetes that results fromdamage to the blood vessels of the light-sensitive tissue at the back ofthe eye (retina). At first, diabetic retinopathy may cause no symptomsor only mild vision problems. Eventually, however, diabetic retinopathycan result in blindness. Diabetic retinopathy can develop in anyone whohas type 1 diabetes or type 2 diabetes.

At its earliest stage, non-proliferative retinopathy, microaneurysmsoccur in the retina's tiny blood vessels. As the disease progresses,more of these blood vessels become damaged or blocked and these areas ofthe retina send signals into the regional tissue to grow new bloodvessels for nourishment. This stage is called proliferative retinopathy.The new blood vessels grow along the retina and along the surface of theclear, vitreous gel that fills the inside of the eye.

By themselves, these blood vessels do not cause symptoms or vision loss.However, they have thin, fragile walls and without timely treatment,these new blood vessels can leak blood (whole blood or a constituentthereof) which can result in severe vision loss and even blindness.

Also, fluid can leak into the center of the macula, the part of the eyewhere sharp, straight-ahead vision occurs. The fluid and the associatedprotein begin to deposit on or under the macula causing the patient'scentral vision to become distorted. This condition is called macularedema. It can occur at any stage of diabetic retinopathy, although it ismore likely to occur as the disease progresses. About half of the peoplewith proliferative retinopathy also have macular edema.

Uveitis is a condition in which the uvea becomes inflamed. The eye isshaped much like a tennis ball, hollow on the inside with threedifferent layers of tissue surrounding a central cavity. The outermostis the sclera (white coat of the eye) and the innermost is the retina.The middle layer between the sclera and the retina is called the uvea.The uvea contains many of the blood vessels that nourish the eye.Complications of uveitis include glaucoma, cataracts or new blood vesselformation (neovascularization).

The currently available interventions for exudative (wet form) maculardegeneration, diabetic retinopathy, diabetic macular edema, choroidalneovascular membrane, complications from uveitis or ocular trauma,include laser photocoagulation therapy, low dose radiation (teletherapy)and surgical removal of neovascular membranes (vitrectomy). Lasertherapy has had limited success and selected choroidal neovascularmembranes which initially respond to laser therapy have high diseaserecurrence rates. There is also a potential loss of vision resultingfrom laser therapy. Low dose radiation has been applied ineffectively toinduce regression of choroidal neovascularization. Recently, vascularendothelial growth factor (VEGF) antagonists, ranibizumab andaflibercept, have been approved for use in age-related maculardegeneration, diabetic macular edema and retinal vein occlusion (RVO).

(RVO) is the most common retinal vascular disease after diabeticretinopathy. Depending on the area of retinal venous drainageeffectively occluded, it is broadly classified as either central retinalvein occlusion (CRVO), hemispheric retinal vein occlusion (HRVO), orbranch retinal vein occlusion (BRVO). It has been observed that each ofthese has two subtypes. Presentation of RVO in general is with variablepainless visual loss with any combination of fundal findings consistingof retinal vascular tortuosity, retinal hemorrhages (blot and flameshaped), cotton wool spots, optic disc swelling and macular edema. In aCRVO, retinal hemorrhages will be found in all four quadrants of thefundus, while these are restricted to either the superior or inferiorfundal hemisphere in a HRVO. In a BRVO, hemorrhages are largelylocalized to the area drained by the occluded in the retinal vein.

There is therefore a long felt and substantial need for methods oftreating diseases of the eye which are characterized by vascularinstability, vascular leakage and neovascularization.

SUMMARY

Disclosed are agents that bind to the extracellular portion and inhibithuman protein tyrosine phosphatase beta (HPTPβ). Also disclosed aremethods for treating eye diseases or conditions characterized byvascular instability, vascular leakage, and neovacularization such asocular edema, ocular neovascularization, diabetic macular edema,age-related macular degeneration, choroidal neovascularization, diabeticretinopathy, retinal vein occlusion (central or branch), ocularischemia, ocular trauma, surgery induced edema, and uveitis.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The monoclonal antibody R15E6 recognizes Endogenous HPTPβ onendothelial cells. (Panel A) Endothelial cell lysates areimmunoprecipitated with a control antibody (Lane 1), with R15E6 (Lane 2)or with a mixture of anti-Tie2 and anti-VEGFR2 antibodies (Lane 3).Immunoprecipitates are resolved by SDS-PAGE, transferred to a PVDFmembrane and probed by western blot with a mixture of R15E6, anti-Tie2and anti-VEGFR2 antibodies. A single major high molecular weight bandconsistent with HPTPβ is seen with R15E6 (Lane 2) and not with thecontrol antibody (Lane 1) or the mixture of anti-Tie2 and anti-VEGFR2(Lane 3). (Panel B) Endothelial cells are subjected to FACS analysiswith R15E6 (white peak) or a control with no primary antibody (blackpeak). The robust shift in fluorescence indicates that R15E6 binds toHPTPβ on the surface of intact endothelial cells.

FIG. 2. The monoclonal antibody R15E6 enhances Tie2 Receptor Activationin HUVECs. Tie2 activation is measured in human endothelial cells asdescribed in Example 4. R15E6 dose dependently enhances both basal andAng1-induced Tie2 activation.

FIG. 3. Is a graphical representation of the mean area of choroidalneovascularization in C57BL/6 mice 14 days post laser injury in eyestreated with intravitreal injection of 1 μg or 2 μg of an anti-VE-PTPextracellular domain antibody in one eye versus similar treatment of thefellow eye with control.

FIG. 4. Shows the mean area (mm²) of retinal neovascularization inC57BL/6 mice on day P17 after containment in a 75% oxygen atmospherefrom P5 to P12 and intravitreal injection of an anti-VE-PTPextracellular domain antibody at P12 when the mice were returned to roomair.

FIG. 5. Shows representative fluorescent micrographs of mouse retinas inthe oxygen-induced retinopathy model after intravitreal injection ofvehicle or 2 μg of an anti-VE-PTP extracellular domain antibody.

FIG. 6. Shows the mean area (mm²) of retinal neovascularization inC57BL/6 mice on day P17 after containment in a 75% oxygen atmospherefrom P5 to P12 followed by return to room air on P12 with subcutaneousadministration of 1 mg/kg of an anti-VE-PTP extracellular domainantibody on days P12, 14 and 16.

FIG. 7. Shows the mean area (mm²) of retinal neovascularization inC57BL/6 mice on day P17 after containment in a 75% oxygen atmospherefrom P5 to P12 followed by return to room air on P12 with subcutaneousadministration of 2 mg/kg of an anti-VE-PTP extracellular domainantibody on days P12, 14 and 16.

DETAILED DESCRIPTION General Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

The term “HPTPβ-ECD binding agent” and “specific binding agent” are usedinterchangeably herein and refer to a molecule that specifically bindsto the extracellular portion of HPTPβ, and variants and derivativesthereof, as defined herein, that inhibits the Tie2 dephosphorylaseactivity of HPTPβ.

“Agent” as used herein refers to a “HPTPβ binding agent” unlessotherwise noted.

“Specifically binds HPTPβ-ECD” refers to the ability of a specificbinding agent of the present invention to recognize and bind to anepitope of the extracellular domain of HPTPβ with higher affinity thanto other related and/or unrelated molecules. Specific binding agentspreferentially bind to HPTPβ in a complex mixture of proteins and/ormacromolecules. The specific binding agent is preferably selective forHPTPβ. “Selective” means that the agent has significantly greateractivity toward HPTPβ compared with other related and/or unrelatedmolecules, not that it is completely inactive with regard to othermolecules. For example, a selective agent may show 10-fold, 100-fold, or1000-fold selectivity toward HPTPβ than to other related or unrelatedmolecules.

The term “anti-HPTPβ-ECD antibodies” refers to antibodies or antibodyfragments that bind to the extracellular domain of HPTPβ. Anti-HPTPβ-ECDantibodies are a type of HPTPβ-ECD binding agent as defined herein.

The term “VE-PTP” refers to the mouse ortholog of HPTPβ.

All percentages, ratios and proportions herein are by weight, unlessotherwise specified. All temperatures are in degrees Celsius (° C.)unless otherwise specified.

Ranges may be expressed herein as from one particular value to anotherparticular value, the endpoints are included in the range. For examplefor the range from “1 mg to 50 mg” includes the specific values 1 mg and50 mg. The antecedent “about” indicates that the values are approximate.For example for the range from “about 1 mg to about 50 mg” indicatesthat the values are approximate values. Additionally, when such a rangeis expressed, the range includes the range “from 1 mg to 50 mg.” It willbe further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. For example the range “from 1 mg to 50 mg”, includesthe range “from 30 mg to 40 mg.”

“Effective amount” means an amount of an active agent or combination ofagents effective to ameliorate or prevent the symptoms, or prolong thesurvival of the patient being treated. An effective amount may varyaccording to factors known in the art, such as the disease state, age,sex and weight of the human or animal being treated. Although particulardosage regimes may be described in examples herein, a person skilled inthe art would appreciate that the dosage regime may be altered toprovide optimum therapeutic response. For example, several divided dosesmay be administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation. In addition,the compositions of this disclosure can be administered as frequently asnecessary to achieve a therapeutic amount. Determination of atherapeutically effective amount is well within the capabilities ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

As used herein the term “inhibit” or “inhibiting” refers to astatistically significant and measurable reduction in activity,preferably a reduction of at least about 10% versus control, morepreferably a reduction of about 50% or more, still more preferably areduction of about 80% or more.

As used herein the term “increase” or “increasing” refers to astatistically significant and measurable increase in activity,preferably an increase of at least about 10% versus control, morepreferably an increase of about 50% or more, still more preferably anincrease of about 80% or more.

“HPTP beta” or “HPTPβ” are used interchangeably herein and areabbreviations for human protein tyrosine phosphatase beta.

As used herein, “subject” means an individual. Thus, the “subject” caninclude domesticated animals (e.g., cats, dogs, etc.), livestock (e.g.,cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g.,mouse, rabbit, rat, guinea pig, etc.) and birds. “Subject” can alsoinclude a mammal, such as a primate or a human. “Subject” and “patient”are used interchangeably herein. Preferably the subject is a human.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,vascular leakage). It is understood that this is typically in relationto some standard or expected value, in other words it is relative, butthat it is not always necessary for the standard or relative value to bereferred to.

The terms “treatment”, “treating”, “treat” and the like, refer toobtaining a desired pharmacologic and/or physiologic effect such asmitigating a disease or a disorder in a host and/or reducing,inhibiting, or eliminating a particular characteristic or eventassociated with a disorder (e.g., ocular edema). Thus, the term“treatment” includes, preventing a disorder from occurring in a host,particularly when the host is predisposed to acquiring the disease, buthas not yet been diagnosed with the disease; inhibiting the disorder;and/or alleviating or reversing the disorder. Insofar as the methods ofthe present invention are directed to preventing disorders, it isunderstood that the term “prevent” does not require that the diseasestate be completely thwarted. Rather, as used herein, the termpreventing refers to the ability of the skilled artisan to identify apopulation that is susceptible to disorders, such that administration ofthe compounds of the present invention may occur prior to onset of adisease. The term does not imply that the disease state is completelyavoided.

Unless otherwise specified, diabetic retinopathy includes all stages ofnon-proliferative retinopathy and proliferative retinopathy.

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a”, “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes one composition or mixtures of two or more such compositions.

Optional” or “optionally” means that the subsequently described event orcircumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

“Specifically binds HPTPβ” refers to the ability of an agent of thepresent invention to recognize and bind to an epitope of theextracellular domain of HPTPβ with higher affinity than to the otherrelated and/or unrelated molecules. The agent is preferably selectivefor HPTPβ. “Specific” means that the agent has significantly greateractivity toward HPTPβ compared with other related and/or unrelatedmolecules, not that it is completely inactive with regard to othermolecules. For example, a selective agent may show 10-fold, 100-fold, or1000-fold selectivity toward HPTPβ than to other related or unrelatedmolecule.

The term “epitope” refers to any portion of any molecule capable ofbeing recognized by and bound by a agent at one or more of the agent'santigen binding regions. Epitopes usually consist of distinct,recognizable surface groupings such as amino acids, sugars, lipids,phosphoryl, or sulfonyl, and, in certain embodiments, may have specificthree dimensional structural characteristics, and/or specific chargecharacteristics. Epitopes as used herein may be conformational orlinear.

“Peptibody” is a molecule comprising an antibody Fc domain attached toat least one peptide. The production of peptibodies is generallydescribed in WO2002/24782.

“Fragment” refers to a portion of an agent. A fragment may retain thedesired biological activity of the agent and may be considered to be anagent itself. For example a truncated protein in which the aminoterminus and/or carboxy terminus and/or an internal amino acid residueis deleted is a fragment of the protein and an Fab of an immunoglobulinmolecule is a fragment of the immunoglobulin. Such fragments may also beconnected to a another molecule by way of a direct connection (e.g. apeptide or disulfide bond) or by way of a linker.

“Protein” is used herein interchangeably with peptide and polypeptide.

Peptides of the present invention include, but are not limited to aminoacid sequences having from about 3 to about 75 amino acids, or fromabout 5 to about 50 amino acids, or from about 10 to about 25 aminoacids. Peptides may be naturally occurring or artificial amino acidsequences.

A protein of the invention may be obtained by methods well known in theart, for example, using standard direct peptide synthesizing techniquessuch as via solid-phase synthesis. If the gene sequence is known or canbe deduced then the protein may be produced by standard recombinantmethods. The proteins may be isolated or purified in a variety of waysknown to one skilled in the art. Standard purification methods includeprecipitation with salts, electrophoretic, chromatographic techniquesand the like.

Agents may be covalently or non-covalently conjugated to a vehicle. Theterm “vehicle” refers to a molecule that prevents degradation and/orincrease half-life, reduces toxicity, reduces immunogenicity, orincreases biological activity of the agent. Exemplary vehicles include,but are not limited, Fc domains of immunoglobulins and polymers, forexample: polyethylene glycol (PEG), polylysine, dextran, a lipid, acholesterol group (such as a steroid); a carbohydrate oroligosaccharide; or any natural or synthetic protein, or peptide thatbinds to a salvage receptor.

“Derivatives” include those binding agents that have been chemicallymodified in some manner distinct from insertion, deletion, orsubstitution variants. For example, wherein the binding agent is aprotein, the carboxyl terminus may be capped with an amino group, suchas NH₂.

In some embodiments one or more molecules are linked together to formthe agent. For example antibody fragments may be connected by a linker.In general, the chemical structure of the linker is not critical as itserves primarily as a space. In one embodiment, the linker is made ofamino acids linked together by way of peptide bonds. In anotherembodiment, the linker is a non-peptide linker such as a non-stericallyhindering C₁-C₆ alkyl group. In another embodiment, the linker is a PEGlinker. It will further be appreciated that the linker can be insertedin a number of locations on the molecule.

Variants of an agent are included within the scope of the presentinvention. “Variant” or “Variants” as used herein means an agent havinga protein or nucleotide sequence which is substantially similar to theprotein or nucleotide sequence of the non-variant agent and which sharesa similar activity of the non-variant agent. A protein or nucleotidesequence may be altered in various ways to yield a variant encompassedby the present invention, including substitutions, deletions,truncations, insertions and other modifications. Methods for suchmanipulations are well known in the art. See, for example, CurrentProtocols in Molecular Biology (and updates) Ausubel et al., Eds (1996),John Wiley and Sons, New York: Methods in Molecular Biology, Vol. 182,In vitro Mutagenesis Protocols, 2^(nd) Edition, Barman Ed. (2002),Humana Press, and the references cited therein. For example, variantsinclude peptides and polypeptides wherein amino acid residues areinserted into, deleted from and/or substituted into the known amino acidsequence for the binding agent. In one embodiment, the substitution ofthe amino acid is conservative in that it minimally alters thebiochemical properties of the variant. In other embodiments, the variantmay be an active fragment of a full-length protein, a chemicallymodified protein, a protein modified by addition of affinity or epitopetags, or fluorescent or other labeling moieties, whether accomplished byin vivo or in vitro enzymatic treatment of the protein, by chemicalmodification, or by the synthesis of the protein using modified aminoacids.

Fusions proteins are also contemplated herein. Using known methods, oneof skill in the art would be able to make fusion proteins of theproteins of the invention; that, while different from native form, maybe useful. For example, the fusion partner may be a signal (or leader)polypeptide sequence that co-translationally or post-translationallydirects transfer of the protein from its site of synthesis to anothersite (e.g., the yeast alpha-factor leader). Alternatively, it may beadded to facilitate purification or identification of the protein of theinvention (e.g., poly-His, Flag peptide, or fluorescent proteins).

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The techniques andprocedures are generally performed according to conventional methodsknown in the art and as described in various general and more specificreferences that are cited and discussed throughout the presentspecification. Unless specific definitions are provided, thenomenclature utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thoseknown and commonly used in the art. Standard techniques may be used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, delivery and treatment of patients.

Sequence Listing

TABLE 1 SEQ ID NO: 1 Full length Human HPTPβ nucleotide sequence(X54131) SEQ ID NO: 2 Full length Human HPTPβ amino acid sequence(P23467) SEQ ID NO: 3 Extracellular Portion of Human HPTPβ with(His)₆Gly Tag SEQ ID NO: 4 Extracellular Portion of Human HPTPβ SEQ IDNO: 5 Full length mouse VE-PTP nucleotide sequence SEQ ID NO: 6 Fulllength mouse VE-PTP amino acid sequence SEQ ID NO: 7 Extracellularportion of mouse VE-PTP amino acid sequence

HPTPβ-ECD Binding Agents

Agents useful in the present invention include, but are not limited to,antibodies, proteins, darpins, peptides, aptamers, adnectins,peptibodies, or nucleic acids that bind specifically to theextracellular portion of HPTPβ and inhibit at least one phosphataseactivity of HPTPβ. As used herein, “phosphatase activity” includesenzymatic activity and biologic activity where biological activity ismeasured by assessing Tie2 phosphorylation.

Agents useful in the present invention further include: antibodies, orantigen binding fragments thereof which bind to the extracellularportion of HPTPβ wherein the antibody or antigen-binding fragmentinhibits at least one phosphatase activity of HPTPβ. These agentsinclude monoclonal and polyclonal antibodies. An agent may be a fragmentof an antibody, wherein the fragment comprises the heavy and light chainvariable regions, or the fragment is an F(ab′)₂, or the fragment is adimer or trimer of an Fab, Fv, scFv, or a dia-, tria-, or tetrabodyderived from the antibody.

For example, the agent may be, without limitation, an antibody orantibody fragment that binds the extracellular portion of HPTPβ; or inparticular an antibody that binds an FN3 repeat of HPTPβ, or morespecifically an antibody that binds the first FN3 repeat of HPTPβ.

Agents further include: the monoclonal antibody R15E6 which is describedin U.S. Pat. No. 7,973,142, which is hereby incorporated in itsentirety. (The mouse hybridoma, Balbc spleen cells (B cells) which maybe used to produce the antibody are deposited with American Type CultureCollection (ATCC), P.O. Box 1549, Manassas, Va. 20108 USA on 4 May 2006,assigned ATCC No. PTA-7580) (Referred to herein as R15E6)), antibodieshaving the same or substantially the same biological characteristics ofR15E6; antibody fragments of R15E6, wherein the fragment comprises theheavy and light chain variable regions; an F(ab′)2 of R15E6; dimers ortrimers of an Fab, Fv, scFv; and dia-, tria-, or tetrabodies derivedfrom R15E6.

In particular, an agent suitable for use in the present invention is anantibody, antibody fragment, variant or derivatives thereof, eitheralone or in combination with other amino acid sequences, provided byknown techniques. Such techniques include, but are not limited toenzymatic cleavage, chemical cleavage, peptide synthesis or recombinanttechniques. The invention further embraces derivative agents, e.g.peptibodies.

Thus, one embodiment of an HPTPβ-ECD binding agent is an antibody,another embodiment is a protein, yet another embodiment is a peptide,and another embodiment is a darpin, another embodiment is an aptamer,another embodiment is a peptibody, still another embodiment is anadnectin, another embodiment is a nucleic acid. In some embodiments theHPTPβ-ECD binding agent is an monoclonal antibody, or is a polyclonalantibody. In particular embodiments, the HPTPβ-ECD binding agent is anantibody fragment that is capable of binding to HPTPβ-ECD. Preferablythe HPTPβ-ECD binding agent is an antibody, or an antibody fragment,including but not limited to, an F(ab′)₂, an Fab, a dimer of an Fab, anFv, a dimer of an Fv, a scFv, a dimer of a scFv, a dimer an Fab, an Fv,a dimer of an Fv, a scFv, a dimer of a scFv, a trimer of an Fab, atrimer of an Fv, a trimer of a scFv, minibodies, a diabody , a triabody,a tetrabody, a linear antibody, a protein, a peptide, an aptamer, apeptibody, an adnectin, or a nucleic acid, that binds to theextracellular portion of HPTPβ. In certain embodiments the HPTPβ-ECDbinding agent is and F(ab′)₂ of a monoclonal antibody. In someembodiments the HPTPβ-ECD binding agent comprises a plurality ofHPTPβ-ECD binding sites, for example where the HPTPβ-ECD binding agentis an intact antibody or an F(ab′)₂, or a dimer of an Fab, or a trimerof an Fab. For example, in some embodiments an HPTPβ-ECD binding agentis able to bind to two HPTPβ molecules simultaneously at the same ordifferent epitope, thereby bringing the two HPTPβ molecules into closeproximity with one and other. In other embodiments the HPTPβ-ECD bindingagent is able to bind to three HPTPβ molecules simultaneously at thesame or different epitope, thereby bringing the three HPTPβ moleculesinto close proximity with one and other. In another embodiment, theHPTPβ-ECD binding agent is the monoclonal antibody produced by hybridomacell line ATCC No. PTA-7680. In yet another embodiment, the HPTPβ-ECDbinding agent is an antigen binding fragment of the monoclonal antibodyproduced by hybridoma cell line ATCC No. PTA-7680. In still anotherembodiment, the HPTPβ-ECD binding agent is an antibody having the sameor substantially the same biological characteristics the monoclonalantibody produced by hybridoma cell line ATCC No. PTA-7680 or an antigenbinding fragment thereof.

Any of the embodiments of HPTPβ-ECD binding agents disclosed in thepresent application, may be covalently or non-covalently conjugated to avehicle. The term “vehicle” refers to a molecule that affects abiological property of an agent. For example, a vehicle may preventdegradation, and/or increase half-life, absorption, reduce toxicity,reduce immunogenicity, or increase biological activity of the agent.Exemplary vehicles include, but are not limited to, Fc domains ofimmunoglobulins; polymers, for example: polyethylene glycol (PEG),polylysine, dextran; lipids; cholesterol groups (such as a steroid);carbohydrates, dendrimers, oligosaccharides, or peptides that binds to asalvage receptor. In some embodiments the vehicle is polyethylene glycol(PEG), in other embodiments the vehicle is polylysine, in yet otherembodiments the vehicle is dextran, in still other embodiments thevehicle is a lipid

Water soluble polymer attachments, such as polyethylene glycol,polyoxyethylene glycol, or polypropylene glycol, as described U.S. Pat.Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and4,179,337, which are incorporated herein in their entirety. Still otheruseful polymers known in the art include monomethoxy-polyethyleneglycol, dextran, cellulose, or other carbohydrate based polymers,poly-(N-vinyl pyrrolidone)-polyethylene glycol, propylene glycolhomopolymers, a polypropylene oxide/ethylene oxide co-polymer,polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol, as wellas mixtures of these polymers. Particularly preferred are peptibodiescovalently modified with polyethylene glycol (PEG) subunits. Watersoluble polymers may be bonded at specific positions, for example at theamino terminus of the peptibodies, or randomly attached to one or moreside chains of the polypeptide. The use of PEG for improving thetherapeutic capacity for agents, e.g. peptibodies, and for humanizedantibodies in particular, is described in U.S. Pat. No. 6,133,426. Theinvention also contemplates derivatizing the peptide and/or vehicleportion of the agents. Such derivatives may improve the solubility,absorption, biological half-life, and the like of the agents. Themoieties may alternatively eliminate or attenuate any undesirableside-effect of the agents and the like.

The term “antibody” (Ab) as used herein includes monoclonal antibodies,polyclonal antibodies, multi-specific antibodies (e.g. bispecificantibodies), single chain antibodies, e.g., antibodies from llama andcamel, antibody fragments, e.g., variable regions and/or constant regionfragments, so long as they exhibit a desired biological activity, e.g.,antigen-binding activity. The term “immunoglobulin” (Ig) is usedinterchangeably with “antibody” herein.

An “antigen binding fragment” as used herein is a fragment of an agentthat binds to a portion of HPTPβ and inhibits the activity of HPTPβ.

An “isolated antibody” is an antibody which has been identified, and/orseparated, and/or recovered from its natural environment.

The basic four-chain antibody unit is a heterotetrameric glycoproteincomposed of two identical light (L) chains and two identical heavy (H)chains (an IgM antibody consists of 5 of the basic heterotetramer unitsalong with an additional polypeptide called J chain, and thereforecontain 10 antigen binding sites, while secreted IgA antibodies maypolymerize to form polyvalent assemblages comprising 2-5 of the basic4-chain units along with J chain). In the case of IgGs, the four-chainunit is generally about 150 kilo Daltons (kDa). Each L chain is linkedto an H chain by one covalent disulfide bond, while the two H chains arelinked to each other by one or more disulfide bonds depending on the Hchain isotype. Each H and L chain also has regularly spaced intrachaindisulfide bridges. Each H chain has at the N-terminus, a variable domain(V_(H)) followed by three constant domains (C_(H)) for each of the alphaand gamma chains and four C_(H) domains for mu and epsilon isotypes.Each L chain has at the N-terminus, a variable domain (V_(L)) followedby a constant domain (C_(L)) at its other end. The V_(L) is aligned withthe V_(H) and the C_(L) is aligned with the first constant domain of theheavy chain (C_(H1)). Particular amino acid residues are believed toform an interface between the light chain and heavy chain variabledomains. The pairing of a V_(H) and V_(L) together forms a singleantigen-binding site. For the structure and properties of the differentclasses of antibodies, see, e.g., Basic and Clinical Immunology, 8thedition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.),Appleton & Lange, 1994, page 71 and Chapter 6.

The L chain from any vertebrate species may be assigned to one of twoclearly distinct types, called kappa and lambda, based on the amino acidsequences of their constant domains. Depending on the amino acidsequence of the constant domain of their heavy chains (C_(H)),immunoglobulins may be assigned to different classes or isotypes. Thereare five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, havingheavy chains designated alpha, delta, epsilon, gamma and mu,respectively. The gamma and alpha classes are further divided intosubclasses on the basis of relatively minor differences in C_(H)sequence and function, e.g., humans express the following subclasses:IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

Members of the Camelidae family, e.g., llama, camel and dromedaries,contain a unique type of antibody, that are devoid of light chains, andfurther lack the C_(H1) domain (Muyldermans, S., Rev. Mol. Biotechnol.,Vol. 74, pp. 277-302 (2001)). The variable region of these heavy chainantibodies are termed V_(HH) or VHH, and constitute the smallestavailable intact antigen-binding fragment (15 kDa) derived from afunctional immunoglobulin.

The term “variable” refers to the fact that certain segments of thevariable domains differ extensively in sequence among antibodies. The Vdomain mediates antigen binding and defines specificity of a particularantibody for its antigen. However, the variability is not evenlydistributed across the 110-amino acid span of the variable domains.Instead, the V regions consist of relatively invariant stretches calledframework regions (FR) of 15-30 amino acids separated by shorter regionsof extreme variability called “hypervariable regions” that are each 9-12amino acids long. The variable domains of native heavy and light chainseach comprise four FRs, largely adopting a β-sheet configuration,connected by three hypervariable regions, which form loops connecting,and in some cases forming part of, the β-sheet structure. Thehypervariable regions in each chain are held together in close proximityby the FRs and, with the hypervariable regions from the other chain,contribute to the formation of the antigen-binding site of antibodies.The constant domains are not involved directly in binding an antibody toan antigen, but exhibit various effector functions, such asparticipation of the antibody in antibody dependent cellularcytotoxicity (ADCC).

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region generally comprises amino acid residues from a“complementarity determining region” or “CDR” (e.g., around aboutresidues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the V_(L), and aroundabout 1-35 (H1), 50-65 (H2) and 95-102 (H3) in the V_(H); Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)) and/orthose residues from a “hypervariable loop”.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. In contrast to polyclonal antibody preparations whichinclude different antibodies directed against different epitopes, eachmonoclonal antibody is directed against a single epitope, i.e., a singleantigenic determinant. In addition to their specificity, the monoclonalantibodies are advantageous in that they may be synthesizeduncontaminated by other antibodies. The modifier “monoclonal” is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies useful in the presentinvention may be prepared by the hybridoma methodology or may be madeusing recombinant DNA methods in bacterial, eukaryotic animal or plantcells (see, e.g., U.S. Pat. No. 4,816,567). The “monoclonal antibodies”may also be isolated from phage antibody libraries, using the availabletechniques, e.g., Clackson et al., Nature, Vol. 352, pp. 624-628 (1991).

The monoclonal antibodies herein include “chimeric” antibodies in whicha portion of the heavy and/or light chain is identical with orhomologous to corresponding sequences in antibodies derived from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is identical with orhomologous to corresponding sequences in antibodies derived from anotherspecies or belonging to another antibody class or subclass, as well asfragments of such antibodies, so long as they exhibit the desiredbiological activity (see U.S. Pat. No. 4,816,567; and Morrison et al.,Proc. Natl. Acad. Sci. USA, Vol. 81, pp. 6851-6855 (1984)).

An “antibody fragment” comprises a portion of a multimeric antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂,dimers and trimers of Fab conjugates, Fv, scFv, minibodies; dia-, tria-and tetrabodies; linear antibodies (See Hudson et al., Nature Med. Vol.9, pp. 129-134 (2003)).

“Fv” is the minimum antibody fragment which contains a complete antigenbinding site. This fragment consists of a dimer of one heavy- and onelight-chain variable region domain in tight, non-covalent association.From the folding of these two domains emanate six hypervariable loops (3loops each from the H and L chain) that contribute the amino acidresidues for antigen binding and confer antigen binding specificity tothe antibody. However, even a single variable domain (or half of an Fvcomprising only three CDRs specific for an antigen) has the ability torecognize and bind antigen, and are therefore included in the definitionof Fv.

A single-chain variable fragment (scFv) is a fusion protein of thevariable regions of the heavy (V_(H)) and light chains (V_(L)) ofimmunoglobulins, connected with a short linker peptide of ten to about25 amino acids. The linker is usually rich in glycine for flexibility,as well as serine or threonine for solubility, and can either connectthe N-terminus of the V_(H) with the C-terminus of the V_(L), or viceversa. This protein retains the specificity of the originalimmunoglobulin, despite removal of the constant regions and theintroduction of the linker.

Divalent (or bivalent) single-chain variable fragments (di-scFvs,bi-scFvs) can be engineered by linking two scFvs. This can be done byproducing a single peptide chain with two V_(H) and two V_(L) regions,yielding tandem scFvs. Another possibility is the creation of scFvs withlinker peptides that are too short for the two variable regions to foldtogether (about five amino acids), forcing scFvs to dimerize. This typeis known as diabodies. Diabodies have been shown to have dissociationconstants up to 40-fold lower than corresponding scFvs, meaning thatthey have a much higher affinity to their target. Consequently, diabodydrugs could be dosed much lower than other therapeutic antibodies andare capable of highly specific targeting of tumors in vivo. Stillshorter linkers (one or two amino acids) lead to the formation oftrimers, so-called triabodies or tribodies. Tetrabodies are known andhave been shown to exhibit an even higher affinity to their targets thandiabodies.

The term “humanized antibody” or “human antibody” refers to antibodieswhich comprise heavy and light chain variable region sequences from anon-human species (e.g., a mouse) but in which at least a portion of theV_(H) and/or V_(L) sequence has been altered to be more “human-like”,i.e., more similar to human germline variable sequences. One type ofhumanized antibody is a CDR-grafted antibody, in which human CDRsequences are introduced into non-human V_(H) and V_(L) sequences toreplace the corresponding nonhuman CDR sequences. Means for makingchimeric, CDR-grafted and humanized antibodies are known to those ofordinary skill in the art (see, e.g., U.S. Pat. Nos. 4,816,567 and5,225,539). One method for making human antibodies employs the use oftransgenic animals, such as a transgenic mouse. These transgenic animalscontain a substantial portion of the human antibody producing genomeinserted into their own genome and the animal's own endogenous antibodyproduction is rendered deficient in the production of antibodies.Methods for making such transgenic animals are known in the art. Suchtransgenic animals may be made using XenoMouse® technology or by using a“minilocus” approach. Methods for making XenoMice® are described in U.S.Pat. Nos. 6,162,963, 6,150,584, 6,114,598 and 6,075,181. Methods formaking transgenic animals using the “minilocus” approach are describedin U.S. Pat. Nos. 5,545,807, 5,545,806, 5,625,825, and WO 93/12227.

Humanization of a non-human antibody has become routine in recent years,and is now within the knowledge of one skilled in the art. Severalcompanies provide services to make a humanized antibody, e.g., Xoma,Aries, Medarex, PDL and Cambridge Antibody Technologies. Humanizationprotocols are extensively described in technical literature, e.g.,Kipriyanov and Le Gall, Molecular Biotechnol, Vol. 26, pp 39-60 (2004),Humana Press, Totowa, N.J.; Lo, Methods Mol. Biol., Vol. 248, pp 135-159(2004), Humana Press, Totowa, N.J.; Wu et al., J. Mol. Biol. Vol. 294,pp. 151-162 (1999).

In certain embodiments, antibodies useful in the present invention maybe expressed in cell lines other than hybridoma cell lines. Sequencesencoding particular antibodies may be used for transformation of asuitable mammalian host cell by known methods for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector), or by transfection proceduresknown in the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461 and 4,959,455. The transformation procedure used may dependupon the host to be transformed. Methods for introduction ofheterologous polynucleotides into mammalian cells are known in the artand include, but are not limited to, dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, mixing nucleic acid withpositively-charged lipids, and direct microinjection of the DNA intonuclei.

A nucleic acid molecule encoding the amino acid sequence of a heavychain constant region, a heavy chain variable region, a light chainconstant region, or a light chain variable region of an antibody, or afragment thereof in a suitable combination if desired, is/are insertedinto an appropriate expression vector using standard ligationtechniques. The antibody heavy chain or light chain constant region maybe appended to the C-terminus of the appropriate variable region and isligated into an expression vector. The vector is typically selected tobe functional in the particular host cell employed (i.e., the vector iscompatible with the host cell machinery such that amplification of thegene and/or expression of the gene may occur). For a review ofexpression vectors, see Methods Enzymol., Vol. 185, (Goeddel, ed.),1990, Academic Press.

Identification of Specific Binding Agents

Suitable HPTPβ-ECD binding agents may be identified using a variety oftechniques known in the art. For example, candidate agents can bescreened for binding to HPTPβ, and screened for activity. Generally, thecandidate agents will first be screened for binding and those that showselective binding will then be screened to determine ability to inhibitthe HPTPβ-mediated dephosphorylation of Tie2. In some cases however thecandidate agents may be first screened in vivo for activity.

Determination of Binding Activity

The selection of a suitable assay for use in identification of aspecific binding agent depends on the nature of the candidate agent tobe screened. One of skill in the art would be able to choose theappropriate assays for the particular candidate agent.

For example, where the candidates are antibodies or peptibodies, whichcomprises an Fc moeity, FACS analysis as described in Example 3B allowsthe candidate agent to be selected based on its ability to bind tocells, which express HPTPβ. The cell may endogenously express HPTPβ ormay be genetically engineered to express HPTPβ.

For other candidate agents such as aptamers, other techniques are knownin the art. For example, aptamers which specifically bind to HPTPβ canbe selected using a technique known as SELEX (systematic evolution ofligands by exponential enrichment) which selects specific aptamersthrough repeated rounds of in vitro selection.

Determination of Inhibitor Activity by Western Blot

As exemplified in Example 4, in one suitable assay HUVECs are culturedin serum free media in the presence or absence of various concentrationsof candidate agent and lysates of the cells are prepared,immunoprecipitated with a Tie2 antibody, resolved by polyacrylamide gelelectrophoresis and transferred to a PVDF membrane. Membrane-boundimmunoprecipitated proteins are then serially western blotted with anantiphosphotyrosine antibody to quantify Tie2 phosphorylation followedby a Tie2 antibody to quantify total Tie2. Tie2 phosphorylation isexpressed as the ratio of the anti-phosphotyrosine signal over the totalTie2 signal. Greater levels of the anti-phosphotyrosine signal indicategreater HPTPβ inhibition by the candidate agent.

Candidate agents that can be screened include, but are not limited to,libraries of known agents, including natural products, such as plant oranimal extracts, biologically active molecules including proteins,peptides including but not limited to members of random peptidelibraries and combinatorial chemistry derived molecular library made ofD- or L-configuration amino acids, antibodies including, but not limitedto, polyclonal, monoclonal, chimeric, human, single chain antibodies,Fab, F(ab)₂ and Fab expression library fragments and eptiope-bindingfragments thereof.

As used herein “antibody fragments” include, but are not limited, to aF(ab′)₂, a dimer or trimer of an Fab, Fv, scFv, or a dia-, tria-, ortetrabody derived from an antibody.

Methods

Disclosed are methods for the treatment of diseases or conditions of theeye, especially retinopathies, ocular edema and ocularneovascularization. Non-limiting examples of these diseases orconditions include diabetic macular edema, age-related maculardegeneration (wet form), choroidal neovascularization, diabeticretinopathy, ocular ischemia, uveitis, retinal vein occlusion (centralor branch), ocular trauma, surgery induced edema, surgery inducedneovascularization, cystoid macular edema, ocular ischemia, uveitis, andthe like. These diseases or conditions are characterized by changes inthe ocular vasculature whether progressive or non-progressive, whether aresult of an acute disease or condition, or a chronic disease orcondition.

One aspect of the disclosed methods relates to diseases that are adirect or indirect result of diabetes, inter alia, diabetic macularedema and diabetic retinopathy. The ocular vasculature of the diabeticbecomes unstable over time leading to conditions such asnon-proliferative retinopathy, macular edema, and proliferativeretinopathy. As fluid leaks into the center of the macula, the part ofthe eye where sharp, straight-ahead vision occurs, the buildup of fluidand the associated protein begin to deposit on or under the macula. Thisresults in swelling that disturbs the subject's central vision. Thiscondition is referred to as “macular edema.” Another condition that mayoccur is non-proliferative retinopathy in which vascular changes, suchas microaneurysms, may occur outside the macular region of the eye.

These conditions may or may not progress to diabetic proliferativeretinopathy which is characterized by neovascularization. These newblood vessels are fragile and are susceptible to bleeding. The result isscaring of the retina, as well as occlusion or total blockage of thelight pathway through the eye due to the over formation of new bloodvessels. Typically, subjects having diabetic macular edema are sufferingfrom the non-proliferative stage of diabetic retinopathy; however, it isnot uncommon for subjects to only begin manifesting macular edema at theonset of the proliferative stage.

Diabetic retinopathy, if left untreated, can lead ultimately toblindness. Indeed, diabetic retinopathy is the leading cause ofblindness in working-age populations.

Therefore, the disclosed methods relate to preventing, treating,controlling, abating, and/or otherwise minimizing ocularneovascularization in a subject having diabetes or a subject diagnosedwith diabetes. In addition, subjects having or subjects diagnosed withdiabetes can be alerted to or can be made aware of the risks ofdeveloping diabetes-related blindness, therefore the present methods canbe used to prevent or delay the onset of non-proliferative retinopathyin subjects known to be at risk Likewise, the present methods can beused for treating subjects having or being diagnosed withnon-proliferative diabetic retinopathy to prevent progression of thecondition.

The disclosed methods relate to preventing or controlling ocularneovascularization or treating a disease or condition that is related tothe onset of ocular neovascularization by administering to a subject aneffective amount of an HPTPβ-ECD binding agent or a pharmaceuticallyacceptable salt thereof.

One aspect of this method relates to treating or preventing ocularneovascularization by administering to a subject an effective amount ofan HPTPβ-ECD binding agent or pharmaceutically acceptable salt thereof.One embodiment of this aspect relates to a method for treating ocularneovascularization comprising administering to a subject a compositioncomprising an effective amount of an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof, and one or more carrier orcompatible excipient.

Thus, one embodiment of the present disclosure is a method of treatingor preventing ocular neovascularization in a subject, comprisingadministering an effective amount of an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure is a method of treating or preventing ocularneovascularization in a subject, comprising administering an effectiveamount of a composition comprising an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof, and one or more carrier orcompatible excipient. Yet another embodiment of the present disclosureis the use of an HPTPβ-ECD binding agent in the treatment of ocularneovascularization.

The disclosed methods also relate to preventing or controlling ocularedema or treating a disease or condition that is related to the onset ofocular edema by administering to a subject an HPTPβ-ECD binding agent.

One aspect of this method relates to treating or preventing ocular edemaby administering to a subject an effective amount of an HPTPβ-ECDbinding agent or a pharmaceutically acceptable salt thereof. Oneembodiment of this aspect relates to a method for treating ocular edemacomprising administering to a subject a composition comprising:

-   -   a. an effective amount of an HPTPβ-ECD binding agent or a        pharmaceutically acceptable salt thereof; and    -   b. one or more carriers or compatible excipients.

Thus, one embodiment of the present disclosure is a method of treatingor preventing ocular edema in a subject, comprising administering aneffective amount of an HPTPβ-ECD binding agent or a pharmaceuticallyacceptable salt thereof. Another embodiment of the present disclosure isa method of treating or preventing ocular edema in a subject, comprisingadministering an effective amount of a composition comprising HPTPβ-ECDbinding agent or a pharmaceutically acceptable salt thereof, and one ormore carriers or compatible excipients. An embodiment of the presentdisclosure is the use of an HPTPβ-ECD binding agent in the treatment ofocular edema.

Another disclosed method relates to preventing or controlling retinaledema or retinal neovascularization, or treating a disease or conditionthat is related to the onset of retinal edema or retinalneovascularization, by administering to a subject an HPTPβ-ECD bindingagent. One aspect of this method relates to treating or preventingretinal edema or retinal neovascularization by administering to asubject an effective amount of an HPTPβ-ECD binding agent orpharmaceutically acceptable salt thereof. One embodiment of this aspectrelates to a method for treating retinal edema or retinalneovascularization comprising administering to a subject a compositioncomprising an effective amount of an HPTPβ-ECD binding agent orpharmaceutically acceptable salt thereof, and one or more carriers orcompatible excipients.

Thus, one embodiment of the present disclosure is a method of treatingor preventing retinal edema in a subject, comprising administering aneffective amount of an HPTPβ-ECD binding agent or a pharmaceuticallyacceptable salt thereof. Another embodiment is a method of treating orpreventing retinal neovascularization comprising administering aneffective amount of an HPTPβ-ECD binding agent or a pharmaceuticallyacceptable salt thereof. One embodiment of the present disclosure is amethod of treating or preventing retinal edema in a subject, byadministering a composition comprising an effective amount of anHPTPβ-ECD binding agent or a pharmaceutically acceptable salt thereof,and one or more carriers or compatible excipients. Another embodiment isa method of treating or preventing retinal neovascularization byadministering an effective amount of an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof, and one or more carriers orcompatible excipients. Another embodiment is the use of an HPTPβ-ECDbinding agent in the treatment of retinal edema. A further embodiment isthe use of an HPTPβ-ECD binding agent in the treatment of retinalneovascularization.

A further disclosed method relates to treating, preventing orcontrolling diabetic retinopathy, or treating a disease or conditionthat is related to the onset of diabetic retinopathy by administering toa subject an HPTPβ-ECD binding agent.

One aspect of this method relates to treating or preventing diabeticretinopathy by administering to a subject an effective amount of anHPTPβ-ECD binding agent or pharmaceutically acceptable salt thereof. Oneembodiment of this aspect relates to a method for treating diabeticretinopathy comprising administering to a subject a compositioncomprising an effective amount of an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof, and one or more carrier orcompatible excipient.

Thus, one embodiment of the present disclosure is a method of treatingor preventing diabetic retinopathy in a subject, comprisingadministering an effective amount of an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure is a method of treating or preventing diabeticretinopathy in a subject, by administering a composition comprising aneffective amount of an HPTPβ-ECD binding agent or a pharmaceuticallyacceptable salt thereof, and one or more carriers or compatibleexcipients. Yet another embodiment of the present disclosure is the useof an HPTPβ-ECD binding agent in the treatment of diabetic retinopathy.

A further disclosed method relates to a method for treating orpreventing non-proliferative retinopathy comprising administering to asubject an effective amount of an HPTPβ-ECD binding agent orpharmaceutically acceptable salt thereof.

Another embodiment of this aspect relates to a method for treating orpreventing non-proliferative retinopathy comprising administering to asubject a composition comprising an effective amount of an HPTPβ-ECDbinding agent or pharmaceutically acceptable salt thereof; and one ormore carrier or compatible excipient.

Thus, one embodiment of the present disclosure is a method of treatingor preventing non-proliferative retinopathy in a subject, comprisingadministering an effective amount of an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure is a method of treating or preventingnon-proliferative retinopathy in a subject, by administering acomposition comprising an effective amount of an HPTPβ-ECD binding agentor a pharmaceutically acceptable salt thereof, and one or more carriersor compatible excipients. Yet another embodiment of the presentdisclosure is the use of an HPTPβ-ECD binding agent in the treatment ofnon-proliferative retinopathy.

Yet a further disclosed method relates to preventing or controllingdiabetic macular edema, or treating a disease or condition that isrelated to the onset of diabetic macular edema by administering to asubject an HPTPβ-ECD binding agent.

One aspect of this method relates to treating or preventing diabeticmacular edema by administering to a subject an effective amount of anHPTPβ-ECD binding agent or pharmaceutically acceptable salt thereof. Oneembodiment of this aspect relates to a method for treating diabeticmacular edema comprising administering to a subject a compositioncomprising: a) an effective amount of one or more of an HPTPβ-ECDbinding agent or a pharmaceutically acceptable salt thereof; and b)oneor more carriers or compatible excipients.

Thus, one embodiment of the present disclosure is a method of treatingor preventing diabetic macular edema in a subject, comprisingadministering an effective amount of an HPTPβ-ECD binding agent or apharmaceutically acceptable salt thereof. Another embodiment of thepresent disclosure is a method of treating or preventing diabeticmacular edema in a subject, by administering a composition comprising aneffective amount of an HPTPβ-ECD binding agent or a pharmaceuticallyacceptable salt thereof, and one or more carriers or compatibleexcipients. Yet another embodiment of the present disclosure is the useof an HPTPβ-ECD binding agent in the treatment of diabetic macularedema.

Another embodiment of the present disclosure is a method for treating,or preventing age-related wet form macular degeneration edema in asubject, comprising administering an effective amount of an HPTPβ-ECDbinding agent or a pharmaceutically acceptable salt thereof. Anotherembodiment of the present disclosure is a method of treating orpreventing age-related wet form macular degeneration edema in a subject,by administering a composition comprising an effective amount of anHPTPβ-ECD binding agent or a pharmaceutically acceptable salt thereof,and one or more carriers or compatible excipients. Yet anotherembodiment of the present disclosure is the use of an HPTPβ-ECD bindingagent in the treatment of age-related wet form macular degenerationedema.

A further embodiment is a method for treating, preventing or controllingchoroidal neovascularization, central retinal vein occlusion, branchretinal vein occlusion, ocular trauma, surgery induced edema, surgeryinduced neovascularization, cystoid macular edema, ocular ischemia, oruveitis, by administering to a subject an effective amount of anHPTPβ-ECD binding agent or a pharmaceutically acceptable salt thereof.Another embodiment is a method for treating, preventing or controllingchoroidal neovascularization, central retinal vein occlusion, branchretinal vein occlusion, ocular trauma, surgery induced edema, surgeryinduced neovascularization, cystoid macular edema, ocular ischemia,retinal angiomatous proliferation, macular telangiectasia, or uveitis,by administering to a subject a composition comprising an effectiveamount of an HPTPβ-ECD binding agent or a pharmaceutically acceptablesalt thereof, and one or more carriers or compatible excipients. Yetanother embodiment of the present disclosure is the use of an HPTPβ-ECDbinding agent in the treatment of choroidal neovascularization, centralretinal vein occlusion, branch retinal vein occlusion, ocular trauma,surgery induced edema, surgery induced neovascularization, cystoidmacular edema, ocular ischemia, retinal angiomatous proliferation,macular telangiectasia or uveitis.

Another embodiment is a composition for treating or preventing an oculardisorder, comprising an HPTPβ-ECD binding agent or pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptablecarrier. Yet another embodiment is a composition for treating orpreventing an ocular disorder, comprising an HPTPβ-ECD binding agent orpharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable carrier composition wherein the oculardisorder is ocular neovascularization, ocular edema, retinalneovascularization, diabetic retinopathy, diabetic macular edema,age-related macular degeneration, choroidal neovascularization, centralretinal vein occlusion, branch retinal vein occlusion, ocular trauma,surgery induced edema, surgery induced neovascularization, cystoidmacular edema, ocular ischemia, non-proliferative retinopathy, retinalangiomatous proliferation, macular telangiectasia, or uveitis.

In some embodiments, the HPTPβ-ECD binding agent or pharmaceuticallyacceptable salt thereof is used for treating an ocular disorder. In someembodiments, the HPTPβ-ECD binding agent or pharmaceutically acceptablesalt thereof is used for treating an ocular disorder, wherein the oculardisorder is ocular neovascularization, ocular edema, retinalneovascularization, diabetic retinopathy, diabetic macular edema,age-related macular degeneration, choroidal neovascularization, centralretinal vein occlusion, branch retinal vein occlusion, ocular trauma,surgery induced edema, surgery induced neovascularization, cystoidmacular edema, ocular ischemia, non-proliferative retinopathy, retinalangiomatous proliferation, macular telangiectasia or uveitis.

In still other embodiments, the HPTPβ-ECD binding agent orpharmaceutically acceptable salt thereof is used for the manufacture ofa medicament for treating an ocular disorder. In some embodiments theocular disorder is ocular neovascularization, ocular edema, retinalneovascularization, diabetic retinopathy, diabetic macular edema,age-related macular degeneration, choroidal neovascularization, centralretinal vein occlusion, branch retinal vein occlusion, ocular trauma,surgery induced edema, surgery induced neovascularization, cystoidmacular edema, ocular ischemia, non-proliferative retinopathy, retinalangiomatous proliferation, macular telangiectasia or uveitis.

Dosing

Effective dosages and schedules for administering the HPTPβ-ECD bindingagent may be determined empirically, and making such determinations iswithin the skill in the art. Those skilled in the art will understandthat the dosage of the agent that must be administered will varydepending on, for example, the subject which will receive the agent, theroute of administration, the particular type of agent used and otherdrugs being administered to the subject. For example, guidance inselecting appropriate doses for antibodies is found in the literature ontherapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies,Ferrone et al., eds., Noges Publications, Park Ridge, N.J., (1985) ch.22 and pp. 303-357; Smith et al., Antibodies in Human Diagnosis andTherapy, Haber et al., eds., Raven Press, New York (1977) pp. 365-389. Atypical dose of the agent used alone might range from about 0.01 mg/kgto up to 500 mg/kg of body weight or more per day, or from about 0.01mg/kg to about 50 mg/kg, or from 0.1 mg/kg to about 50 mg/kg, or fromabout 0.1 mg/kg to up to about 10 mg/kg, or from about 0.2 mg/kg toabout 1 mg/kg, depending on the factors mentioned above.

One embodiment relates to a method for treating ocular edema and/orneovascularization comprising administering to a subject from about 0.01mg/kg to about 50 mg/kg of an HPTPβ-ECD binding agent orpharmaceutically acceptable salt thereof. Another iteration of thisembodiment relates to administering to a subject from about 0.1 mg/kg toabout 10 mg/kg by weight of the subject being treated, an HPTPβ-ECDbinding agent or pharmaceutically acceptable salt thereof. A furtheriteration of this embodiment relates to a method for treating orpreventing diseases or conditions related to ocular edema and/orneovascularization comprising administering to a subject from about 1mg/kg to about 10 mg/kg by weight of the subject an HPTPβ-ECD bindingagent or pharmaceutically acceptable salt thereof. Yet another iterationof this embodiment relates to a method for treating or preventingdiseases or conditions related to ocular edema and/or neovascularizationcomprising administering to a subject from about 5 mg/kg to about 10mg/kg by weight of the subject an HPTPβ-ECD binding agent orpharmaceutically acceptable salt thereof. In a further iteration of thisembodiment relates to a method for treating or preventing diseases orconditions related to ocular edema and/or neovascularization comprisingadministering to a subject from about 1 mg/kg to about 5 mg/kg by weightof the subject an HPTPβ-ECD binding agent or pharmaceutically acceptablesalt thereof. In a yet further iteration of this embodiment relates to amethod for treating or preventing diseases or conditions related toocular edema and/or neovascularization comprising administering to asubject from about 3 mg/kg to about 7 mg/kg by weight of the subject anHPTPβ-ECD binding agent or pharmaceutically acceptable salt thereof.

The dosing schedules for administration of an HPTPβ-ECD binding agentinclude, but are not limited to, once daily, three-times weekly, twiceweekly, once weekly, three times, twice monthly, once monthly and onceevery other month.

Further disclosed are methods of treating or preventing one or more ofthe diseases or conditions described herein above related to ocularedema and/or neovascularization that are the result of administration ofanother pharmaceutically active agent. As such, this aspect relates to amethod comprising administering to a subject a composition comprising:a) an effective amount of an HPTPβ-ECD binding agent or pharmaceuticallyacceptable salt thereof; b) one or more additional pharmaceuticallyactive agents; and c) one or more carriers or compatible excipients.

The methods of the present invention may be combined with the standardof care, including, but not limited to, laser treatment.

Non-limiting examples of pharmaceutically active agents suitable forcombination with an HPTPβ-ECD binding agent include anti-infectives,i.e., aminoglycosides, antiviral agents, antimicrobials,anticholinergics/antispasmotics, antidiabetic agents, antihypertensiveagents, antineoplastics, cardiovascular agents, central nervous systemagents, coagulation modifiers, hormones, immunologic agents,immunosuppressive agents, ophthalmic preparations and the like.

The disclosed method also relates to the administration of the disclosedagents and compositions. Administration can be systemic via subcutaneousor i.v. administration; or the HPTP-β inhibitor will be administereddirectly to the eye, e.g., local. Local methods of administrationinclude, for example, by eye drops, subconjunctival injections orimplants, intravitreal injections or implants, sub-Tenon's injections orimplants, incorporation in surgical irrigating solutions, etc.

The disclosed methods relate to administering an HPTPβ-ECD binding agentas part of a pharmaceutical composition. Compositions suitable for localadministration are known to the art (see, for example, U.S. Pat. Publ.2005/0059639). In various embodiments, compositions of the invention cancomprise a liquid comprising an active agent in solution, in suspension,or both. As used herein, liquid compositions include gels. In oneembodiment, the liquid composition is aqueous. Alternatively, thecomposition can take form of an ointment. In another embodiment, thecomposition is an in situ gellable aqueous composition. Such acomposition can comprise a gelling agent in a concentration effective topromote gelling upon contact with the eye or lacrimal fluid in theexterior of the eye. Aqueous compositions of the invention haveophthalmically compatible pH and osmolality. The composition cancomprise an ophthalmic depot formulation comprising an active agent forsubconjunctival administration. The microparticles comprising activeagent can be embedded in a biocompatible pharmaceutically acceptablepolymer or a lipid encapsulating agent. The depot formulations may beadapted to release all or substantially all the active material over anextended period of time. The polymer or lipid matrix, if present, may beadapted to degrade sufficiently to be transported from the site ofadministration after release of all or substantially all the activeagent. The depot formulation can be a liquid formulation, comprising apharmaceutical acceptable polymer and a dissolved or dispersed activeagent. Upon injection, the polymer forms a depot at the injections site,e.g., by gelifying or precipitating. The composition can comprise asolid article that can be inserted in a suitable location in the eye,such as between the eye and eyelid or in the conjuctival sac, where thearticle releases the active agent. Solid articles suitable forimplantation in the eye in such fashion generally comprise polymers andcan be bioerodible or non-bioerodible.

In one embodiment of the disclosed methods, a human subject with atleast one visually impaired eye is treated with 2-4000 μg of anHPTPβ-ECD binding agent via intravitreal injection. Improvement ofclinical symptoms are monitored by one or more methods known to the art,for example, indirect ophthalmoscopy, fundus photography, fluoresceinangiopathy, electroretinography, external eye examination, slit lampbiomicroscopy, applanation tonometry, pachymetry, optical coherencetomography and autorefaction. Subsequent doses can be administeredweekly or monthly, e.g., with a frequency of 2-8 weeks or 1-12 monthsapart.

The disclosed methods include administration of the disclosed agents incombination with a pharmaceutically acceptable carrier.“Pharmaceutically acceptable” means a material that is not biologicallyor otherwise undesirable, i.e., the material may be administered to asubject without causing any undesirable biological effects orinteracting in a deleterious manner with any of the other components ofthe pharmaceutical formulation in which it is contained. The carrierwould naturally be selected to minimize any degradation of the activeingredient and to minimize any adverse side effects in the subject, aswould be well known to one of skill in the art. In another aspect, manyof the disclosed agents can be used prophylactically, i.e., as apreventive agent, either neat or with a pharmaceutically acceptablecarrier. The ionic liquid compositions disclosed herein can beconveniently formulated into pharmaceutical compositions composed ofneat ionic liquid or in association with a pharmaceutically acceptablecarrier. See Remington's Pharmaceutical Sciences, 18th ed., Gennaro, AR. Ed., Mack Publishing, Easton Pa. (1990), which discloses typicalcarriers and conventional methods of preparing pharmaceuticalcompositions that can be used in conjunction with the preparation offormulations of the agents described herein and which is incorporated byreference herein. Such pharmaceutical carriers, most typically, would bestandard carriers for administration of compositions to humans andnon-humans, including solutions such as sterile water, saline andbuffered solutions at physiological pH. Other agents can be administeredaccording to standard procedures used by those skilled in the art. Forexample, pharmaceutical compositions can also include one or moreadditional active ingredients such as antimicrobial agents,anti-inflammatory agents, anesthetics and the like.

Examples of pharmaceutically-acceptable carriers include, but are notlimited to, saline, Ringer's solution and dextrose solution. The pH ofthe solution is preferably from about 5 to about 8, and more preferablyfrom about 7 to about 7.5. Further carriers include sustained releasepreparations such as semipermeable matrices of solid hydrophobicpolymers containing the disclosed agents, which matrices are in the formof shaped articles, e.g., films, liposomes, microparticles, ormicrocapsules. It will be apparent to those persons skilled in the artthat certain carriers can be more preferable depending upon, forinstance, the route of administration and concentration of compositionbeing administered. Other agents can be administered according tostandard procedures used by those skilled in the art.

Pharmaceutical formulations can include additional carriers, as well asthickeners, diluents, buffers, preservatives, surface active agents andthe like in addition to the agents disclosed herein. Pharmaceuticalformulations can also include one or more additional active ingredientssuch as antimicrobial agents, anti-inflammatory agents, anesthetics andthe like.

For the purposes of the present disclosure the term “excipient” and“carrier” are used interchangeably throughout the description of thepresent disclosure and said terms are defined herein as, “ingredientswhich are used in the practice of formulating a safe and effectivepharmaceutical composition.”

The formulator will understand that excipients are used primarily toserve in delivering a safe, stable and functional pharmaceutical,serving not only as part of the overall vehicle for delivery but also asa means for achieving effective absorption by the recipient of theactive ingredient. An excipient may fill a role as simple and direct asbeing an inert filler, or an excipient as used herein may be part of apH stabilizing system. The formulator can also take advantage of thefact the agents of the present invention have improved cellular potency,pharmacokinetic properties.

The disclosed agents can also be present in liquids, emulsions, orsuspensions for delivery of active therapeutic agents. Liquidpharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc., an active agent as describedherein and optional pharmaceutical adjuvants in an excipient, such as,for example, water, saline aqueous dextrose, glycerol, ethanol and thelike, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered can also contain minoramounts of nontoxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents and the like, for example, sodium acetate,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, etc. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in this art, for example seeRemington's Pharmaceutical Sciences, referenced above.

Kits

Also disclosed are kits comprising the agents and compositions to bedelivered into a human, mammal, or cell. The kits can comprise one ormore packaged unit doses of a composition comprising one or more agentsto be delivered into a human, mammal, or cell. The unit dosage ampoulesor multi-dose containers, in which the agents to be delivered arepackaged prior to use, can comprise a hermetically sealed containerenclosing unit dose of the composition, or multiples unit doses. Theagents can be packaged as a sterile formulation, and the hermeticallysealed container is designed to preserve sterility of the formulationuntil use.

EXAMPLES Example 1 Production of HPTPβ Extracellular Domain Protein

Full length HPTPβ cDNA (SEQ ID NO:1) is cloned from a human placentallibrary according to the manufacturer's (Origene) instructions. A cDNAencoding the entire soluble extracellular domain (ECD) of HPTPβ iscloned by PCR from the full length cDNA coding for amino acids 1-1621with an added c-terminal His-His-His-His-His-His-Gly (6His-Gly) (SEQ IDNO:3). The resulting cDNA is cloned into mammalian expression vectorsfor transient (pShuttle-CMV) or stable (pcDNA3.1(−)) expression inHEK293 cells. To obtain purified HPTPβ ECD (βED), HEK293 cellstransfected with a βECD expression vector are incubated in OptiMEM-serumfree (Gibco) for 24 hours under normal growth conditions. Theconditioned media is then recovered, centrifuged to remove debris, and 1mL of washed Ni-NTA agarose (Qiagen) (500 μL packed material) is addedto each 10 μL of cleared media and allowed to rock overnight at 4° C. Onthe following day, the mixture is loaded into a column and washed with20 bed volumes of 50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole, pH 8. Thepurified HPTPβ extracellular domain protein (SEQ ID NO:4) is then elutedwith 200 μL/elution in 50 mM NaH₂PO₄, 300 mM NaCl, 250 mM Imidazole, pH8. Fractions are analyzed for protein content using reducing-denaturingSDS-polyacrylimide gel electrophoresis and detected by silver stain(Invitrogen) and confirmed by mass spectrometry.

Example 2 Generation of Monoclonal Antibodies to HPTPβ ExtracellularDomain

Purified HPTPβ extracellular domain protein is produced, for example bythe procedure described in Example 1. For production of the HPTPβextracellular domain immunogen, the purified HPTPβ extracellulardomain-6-His protein is conjugated to porcine thyroglobulin (Sigma)using EDC coupling chemistry (Hockfield, S. et al., (1993) Cold SpringHarbor Laboratory Press. Vol. 1 pp. 111-201, Immunocytochemistry). Theresulting HPTPβ extracellular domain-thyroglobulin conjugate is dialyzedagainst PBS, pH 7.4. Adult Balb/c mice are then immunized subcutaneouslywith the conjugate (100-200 μg) and complete Freund's adjuvant in a 1:1mixture. After 2-3 weeks, the mice are injected intraperitoneally orsubcutaneously with incomplete Freund's adjuvant and the conjugate in a1:1 mixture. The injection is repeated at 4-6 weeks. Sera are collectedfrom mice 7 days post-third-injection and assayed for immunoreactivityto HPTPβ extracellular domain antigen by ELISA and western blotting.Mice that display a good response to the antigen are boosted by a singleintra-spleen injection with 50 μl of purified HPTPβ extracellular domainprotein mixed 1:1 with Alum hydroxide using a 31 gauge extra long needle(Goding, J. W., (1996) Monoclonal Antibodies: Principles and Practices.Third Edition, Academic Press Limited. p. 145). Briefly, mice areanesthetized with 2.5% avertin, and a 1 centimeter incision is createdon the skin and left oblique body wall. The antigen mixture isadministered by inserting the needle from the posterior portion to theanterior portion of the spleen in a longitudinal injection. The bodywall is sutured and the skin is sealed with two small metal clips. Miceare monitored for safe recovery. Four days after surgery the mousespleen is removed and single cell suspensions are made for fusion withmouse myeloma cells for the creation of hybridoma cell lines (Spitz, M.,(1986) Methods In Enzymology, Vol. 121. Eds. John J, Lagone and HelenVan Vunakis. pp. 33-41 (Academic Press, New York, N.Y.)). Resultinghybridomas are cultured in Dulbeccos modified media (Gibco) supplementedwith 15% fetal calf serum (Hyclone) and hypoxathine, aminopterin andthymidine.

Screening for positive hybridomas begins 8 days after the fusion andcontinues for 15 days. Hybridomas producing anti-HPTPβ extracellulardomain antibodies are identified by ELISA on two sets of 96-well plates:one coated with the histidine tagged-HPTPβ extracellular domain andanother one coated with a histidine-tagged bacterial MurA protein as anegative control. The secondary antibody is a donkey anti-mouse IgGlabeled with horseradish peroxidase (HRP) (Jackson Immunoresearch).Immunoreactivity is monitored in wells using color development initiatedby ABTS tablets dissolved in TBS buffer, pH 7.5. The individual HRPreaction mixtures are terminated by adding 100 microliters of 1% SDS andreading absorbance at 405 nm with a spectrophotometer. Hybridomasproducing antibodies that interact with HPTPβ extracellular domain-6His,and not with the murA-6His protein are used for further analysis.Limiting dilutions (0.8 cells per well) are performed twice on positiveclones in 96 well plates, with clonality defined as having greater than99% of the wells with positive reactivity. Isotypes of antibodies aredetermined using the iso-strip technology (Roche). To obtain purifiedantibody for further evaluation, tissue culture supernatants areaffinity purified using a protein A or protein G columns.

Five monoclonal antibodies immunoreactive to HPTPβ-ECD protein wereisolated and given the following nomenclature, R15E6, R12A7, R3A2,R11C3, R15G2 and R5A8. Based on its reaction with the HPTPβ-ECD proteinin ELISA and in western blots, R15E6 was selected for further study.

Example 3 The Monoclonal Antibody R15E6

The monoclonal antibody R15E6 was identified and characterized asdescribed in Example 2 of the present application and in U.S. Pat. No.7,973,142; the procedure and results are summarized below.

A. R15E6 Binds Endogenous HPTPβ as Demonstrated by Demonstrated byImmunoprecipitation.

Materials: Human umbilical vein endothelial cells (HUVECs), EGM media,and trypsin neutralizing solution from Cambrex; OPTIMEM I (Gibco),bovine serum albumin (BSA; Santa Cruz), phosphate buffered saline (PBS;Gibco), Growth Factors including Angiopoietin 1 (Ang1), vascularendothelial growth factor (VEGF) and fibroblast growth factor (FGF) (R&DSystems), Tie2 monoclonal antibody (Duke University/P&GP), VEGF receptor2 (VEGFR2) polyclonal antibody (Whitaker et. al), protein A/G agarose(Santa Cruz), Tris-Glycine pre-cast gel electrophoresis/transfer system(6-8%) (Invitrogen), PVDF membranes (Invitrogen), lysis buffer (20 mmTris-HCl, 137 mm NaCl, 10% glycerol, 1% triton-X-100, 2 mM EDTA, 1 mMNaOH, 1 mM NaF, 1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin).

Method: HUVECs were pre-treated for 30 min with antibody (in OPTIMEM) orOPTIMEM I alone. After removal of pre-treatment, cells were treated withAng1 (100 ng/ml) for 6 minutes in PBS+0.2% BSA and lysed in lysisbuffer. Lysates were run directly on a Tris-Glycine gel orimmunoprecipitated with 2-5 μg/ml Tie-2 antibody or 10 μg/ml R15E6antibody and protein A/G agarose. Immunoprecipitated samples were rinsedonce with lysis buffer and boiled for 5 min in 1× times sample buffer.Samples were resolved on a Tris-Glycine gel, transferred to a PVDFmembrane, and detected by western blot using the indicated antibodies(pTYR Ab (PY99, Santa Cruz), Tie-2, VEGFR2 and/or R15E6).

Results: By IP/western blotting, R15E6 recognizes a major, highmolecular weight band consistent with the size of HPTPβ (FIG. 1, PanelA, Lane 2). The less intense, lower molecular weight bands likelyrepresent less glycosylated precursor forms of HPTPβ. Animmunoprecipitation (IP) with control, non-immune IgG shows no bands inthe molecular weight range of HPTPβ (FIG. 1, Panel A, Lane 1), and acombined Tie2/VEGFR2 IP shows bands of the expected molecular weight(FIG. 1, Panel A, Lane 3). This result demonstrates that R15E6recognizes and is specific for HPTPβ.

B. R15E6 Binds Endogenous HPTPβ as Demonstrated by FACS Analysis

Materials: HUVECs, EGM media, and trypsin neutralizing solution fromCambrex; Secondary Alexfluor 488-tagged antibody from Molecular Probes;Hanks balanced salt solution (Gibco); FACSCAN flow cytometer andCellQuest software from Becton Dickenson.

Method: HUVECs are trypsinized, treated with trypsin neutralizingsolution and rinsed with HBSS. R15E6 antibody (0.6 μg) is added to250,000 cells in 500 of HBSS and incubated on ice for 20 minutes. Cellswere rinsed with 1 ml HBSS followed by adding 2 μg offluorescent-conjugated secondary antibody for 20 minutes on ice. Cellswere rinsed and resuspended in 1 ml HBSS then analyzed on the FACSCANflow cytometer with CellQuest software. Control cells were treated withfluorescent-conjugated secondary antibody only.

Results: By FACS analysis, intact HUVECs, R15E6 causes a robust shift(>90% of cells) in the fluorescence signal compared to the secondaryantibody alone (FIG. 1, Panel B). This result indicates that R15E6 bindsto endogenous HPTPβ presented on the surface of intact endothelialcells.

Example 4 R15E6 Enhances Tie2 Activation

R15E6 enhances Tie2 phosphorylation in the absence and presence of theangiopoietin 1 (Ang1), the Tie2 ligand.

Methods: HUVECs are cultured in serum free media as described above inthe presence or absence of various concentrations of R15E6 and with orwithout added Ang1. Lysates are prepared, immunoprecipitated with a Tie2antibody, resolved by polyacrylamide gel electrophoresis and transferredto a PVDF membrane. Membrane-bound immunoprecipitated proteins are thenserially western blotted with an antiphosphotyrosine antibody toquantify Tie2 phosphorylation followed by a Tie2 antibody to quantifytotal Tie2. Tie2 phosphorylation is expressed as the ratio of theantiphosphotyrosine signal over the total Tie2 signal.

Results: R15E6 enhances Tie2 phosphorylation both in the absence andpresence of Ang1 (FIG. 2). This result indicates that binding of R15E6to HPTPβ on the surface of endothelial cells modulates its biologicalfunction resulting in enhanced activation of Tie2 in the absence orpresence of ligand.

Example 5 Generation of Anti-VE-PTP Extracellular Domain Antibodies A.Production of Mouse VE-PTP Extracellular Domain Protein (VE-PTP-ECD)

VE-PTP-ECD may be produced by any suitable method. Such methods are wellknown in the art. For example, VE-PTP-ECD can be produced using a methodsimilar to Example 1 of the present disclosure where VE-PTP-ECD cDNA isused in place of cDNA encoding HPTPβ-ECD. SEQ ID NO:7 provides anucleotide sequence that encodes VE-PTP-ECD. SEQ ID NO:8 provides theamino acid sequence of VE-PTP-ECD.

B. Generation of Antibodies to VE-PTP ECD

Anti-VE-PTP antibodies are readily generated by methods that are wellknown in the art. For example, anti VE-PTP antibodies can be generatedusing the method of Example 2 of the present disclosure by substitutingVE-PTP-ECD for the HPTPβ extracellular domain and immunizing rats withthe resulting protein. The rat anti-mouse VE-PTP antibody used in thepresent studies was kindly provided by Dr. D. Vestweber (mAb 109). Theantibody was generated as described in Baumer S. et al., Blood, 2006;107: 4754-4762. Briefly, the antibody was generated by immunizing ratswith a VE-PTP-Fc fusion protein. Immunization, hybridoma-fusion, andscreening were conducted as described in Gotsch U., et al., J Cell Sci.1997, Vol. 110, pp. 583-588 and Bosse R. and Vestweber D., Eur JImmunol. 1994, Vol. 24, pp. 3019-3024.

The fusion protein was constructed such that the first 8 fibronectintype III-like repeats ending with the amino acid proline at position 732of VE-PTP were fused in frame with the Fc part of human IgG1 (startingwith amino acid proline at position 239). This construct cloned intopcDNA3 (Invitrogen) was stably transfected into CHO cells, and thefusion protein was purified by protein A Sepharose affinitypurification.

Example 6 Intravitreal Injections of an Anti-VE-PTP ECD Antibody

Laser-induced Choroidal Neovascularization Model: The choroidalneovascularization model is considered to represent a model ofneovascular age-related macular degeneration. Choroidal NV was generatedas previously described. See Tobe T, et al., Am. J. Pathol. 1998, Vol.153, pp. 1641-1646. Adult C57BL/6 mice had laser-induced rupture ofBruch's membrane in three locations in each eye and were then given 1 μLintravitreal injections of 1 or 2 μg of a VE-PTP-ECD antibody (IgG2a),in one eye and vehicle (5% dextrose) in the fellow eye. These treatmentswere repeated on day 7. Fourteen days after laser, the mice wereperfused with fluorescein-labeled dextran (2×10⁶ average MW, Sigma, St.Louis, Mo.) and the extent of neovascularization was assessed inchoroidal flat mounts by fluorescence microscopy. The area of CNV ateach Bruch's membrane rupture site was measured by image analysis by anobserver masked with respect to treatment group. The area of CNV is theaverage of the three rupture sites in one eye. As shown in FIG. 3,treatment with the VE-PTP-ECD antibody significantly reduced choroidalneovascularization at both 1 and 2 μg doses versus treatment withvehicle control.

Example 7 Oxygen-Induced Ischemic Retinopathy

The oxygen-induced ischemic retinopathy model is considered to representa model of proliferative diabetic retinopathy. Ischemic retinopathy wasproduced in C57BL/6 mice by a method described by Smith, L. E. H., etal. Oxygen-induced retinopathy in the mouse. Invest. Ophthalmol. Vis.Sci. 35, 101-111 (1994).

C57BL/6 mice at postnatal day 7 (P7) and their mothers were placed in anairtight chamber and exposed to hyperoxia (75±3% oxygen) for five days.Oxygen was continuously monitored with a PROOX model 110 oxygencontroller (Reming Bioinstruments Co., Redfield, N.Y.). On P12, micewere returned to room air and under a dissecting microscope, a HarvardPump Microinjection System and pulled glass pipettes were used todeliver a 1 μl intravitreal injection of 1 or 2 μg of a VE-PTP-ECDantibody was made in one eye and vehicle was injected in the fellow eye.At P17, the area of NV on the surface of the retina was measured at P17as previously described. See Shen J, et al., Invest. Ophthalmol. Vis.Sci. 2007, Vol. 48, pp. 4335-4341. Briefly, mice were given anintraocular injection of 1 μl containing 0.5 μg rat anti-mouse PECAMantibody (Pharmingen, San Jose, Calif.). Twelve hours later, the micewere euthanized, the eyes fixed in 10% formalin. The retinas weredissected, incubated for 40 minutes in 1:500 goat anti-rat IgGconjugated with Alexa488 (Invitrogen, Carlsbad, Calif.), washed, andwhole mounted. An observer masked with respect to treatment groupexamined the slides with a Nikon Fluorescence microscope and measuredthe area of NV per retina by computerized image analysis using ImageProPlus software (Media Cybernetics, Silver Spring, Md.). FIG. 4 shows thattreatment with the VE-PTP-ECD antibody significantly reduced retinalneovascularization at both 1 and 2 μg doses versus treatment withvehicle control. FIG. 5 shows representative retinal whole mounts from amouse treated with vehicle versus a mouse treated with 2 μg of theVE-PTP-ECD antibody.

Example 8 Subcutaneous Injection of a VE-PTP-ECD Antibody

The oxygen-induced ischemic retinopathy model was conducted as describedin Example 7 (containment in a 75% oxygen atmosphere from P5 to P12) forintravitreal dosing except that the VE-PTP-ECD antibody (1 mg/kg) wasdosed subcutaneously at P12 when the mice were returned to room air andagain on days P14 and P16 (three total doses). Neovascularization wasassessed as described above on day (P17). FIG. 6 shows that subcutaneousdosing of the VE-PTP-ECD antibody reduces the area of retinalneovascularization.

Example 9

The experiment described in Example 8 was repeated at a subcutaneousdose of 2 mg/kg. (FIG. 7)

While a number of embodiments of this disclosure are described, it isapparent that the basic examples may be altered to provide otherembodiments that utilize or encompass the HPTPβ-ECD binding agent,methods and processes of this invention. The embodiments and examplesare for illustrative purposes and are not to be interpreted as limitingthe disclosure, but rather, the appended claims define the scope of thisinvention.

1-20. (canceled)
 21. A pharmaceutical composition comprising atherapeutically-effective amount of a multi-specific antibody, whereinthe multi-specific antibody binds to a human protein tyrosinephosphatase beta-extracellular domain (HPTPβ-ECD) in a subject, andwherein the therapeutically-effective amount of the multi-specificantibody is from about 0.01 mg/kg to about 500 mg/kg by weight of thesubject.
 22. The pharmaceutical composition of claim 21, wherein themultispecific antibody binds to an FN3 repeat in the HPTPβ-ECD.
 23. Thepharmaceutical composition of claim 21, wherein the multispecificantibody binds to a first FN3 repeat in the HPTPβ-ECD.
 24. Thepharmaceutical composition of claim 21, wherein the multispecificantibody inhibits HPTPβ.
 25. The pharmaceutical composition of claim 21,wherein the multispecific antibody binds to SEQ ID NO.:
 4. 25. Thepharmaceutical composition of claim 21, wherein the pharmaceuticalcomposition is a solution, and the solution has a pH of from about 7 toabout 7.5.
 26. The pharmaceutical composition of claim 21, wherein themulti-specific antibody is a bispecific antibody.
 27. The pharmaceuticalcomposition of claim 21, wherein the therapeutically-effective amount ofthe multi-specific antibody is from about 0.1 mg/kg to about 50 mg/kg.28. The pharmaceutical composition of claim 21, further comprising acarrier.
 29. A method for treating an ocular condition in a subject inneed thereof, the method comprising administering to the subject apharmaceutical composition, wherein the pharmaceutical compositioncomprises a therapeutically-effective amount of a multi-specificantibody, wherein the multi-specific antibody binds to a human proteintyrosine phosphatase beta-extracellular domain (HPTPβ-ECD) in a subject,and wherein the therapeutically-effective amount of the multi-specificantibody is from about 0.01 mg/kg to about 500 mg/kg by weight of thesubject.
 30. The method of claim 29, wherein the multispecific antibodybinds to an FN3 repeat in the HPTPβ-ECD.
 31. The method of claim 29,wherein the multispecific antibody binds to a first FN3 repeat in theHPTPβ-ECD.
 32. The method of claim 29, wherein the multispecificantibody inhibits HPTPβ.
 33. The method of claim 29, wherein themultispecific antibody binds to SEQ ID NO.:
 4. 34. The method of claim29, wherein the pharmaceutical composition is a solution, and thesolution has a pH of from about 7 to about 7.5.
 35. The method of claim29, wherein the multi-specific antibody is a bispecific antibody. 36.The method of claim 29, wherein the therapeutically-effective amount ofthe multi-specific antibody is from about 0.1 mg/kg to about 50 mg/kg.37. The method of claim 29, wherein the pharmaceutical composition isadministered by intraocular injection.
 38. The method of claim 29,wherein the pharmaceutical composition is administered by subcutaneousinjection.
 39. The method of claim 29, wherein the pharmaceuticalcomposition is administered by intravenous injection.
 40. The method ofclaim 29, wherein the subject is human.
 41. The method of claim 29,wherein the pharmaceutical composition further comprises a carrier.